Introduction. Infections of the bloodstream and central nervous system (CNS) caused by Pseudomonas aeruginosa are associated with a serious patient conditions and are often accompanied by high mortality.Aim. Molecular genetic characterization of P. aeruginosa isolated from positive samples of blood cultures and cerebrospinal fluid of patients under 18 years of age from intensive care units of hospitals.Materials and methods. We conducted a retrospective study of bacteremia and CNS infection cases associated with P. aeruginosa from 2014 to 2021. 24 clinical isolates of P. aeruginosa from positive blood cultures and CSF were analyzed. MICs of antibiotics were determined by serial microdilution in broth. Identification of the genes of carbapenemase was carried out using real-time PCR. Virulence genes were determined by PCR. Population diversity was assessed by MLST.Results. More than 70% of isolates showed resistance to carbapenem antibiotics. The phenotype of multiple drug resistance had 25% of the isolates. Extreme resistance was shown by 54% of isolates. The detection rate of metallo-β-lactamases (MBL) was 54%. Based on PCR data, 33% of the strains were found to have the ExoU type, and 67% had the ExoS type. According to MLST, 16 genotypes were identified. The structure was dominated by two sequence types ST654 (29%) and ST235 (12.5%). The structure of patients was dominated by children with surgical pathology — 16 cases, and there were eight somatic patients. Fatal outcome was observed in 28% of cases with bacteremia and CNS infection associated with P. aeruginosa.Conclusion. P. aeruginosa isolates from positive blood cultures and CSF samples are highly resistant to antibiotics; virulence genes were found in all isolates. Strains of high epidemic risk prevailed in the studied sample. More than a quarter of the described clinical cases had an unfavorable outcome.

Acinetobacter baumannii is a representative of the peak priority nosocomial pathogens capable of causing infections with high mortality and economic treatment costs. The purpose of our study was to determine a role of A. baumannii in blood-borne and central nervous system infections in children. We conducted a retrospective study of A. baumannii associated cases of bacteremia and CNS infection in children. A. baumannii strains were isolated from 17 children followed up with surgical pathology (congenital heart defects 24%, abdominal pathology 29%, severe combined trauma 29%) and with somatic diseases accompanied by antibacterial and/or glucocorticosteroid therapy 18%. The minimum inhibitory concentrations of antibiotics were determined by the broth microdilution method. Carbapenemase genes were detected by real time polymerase chain reaction. Biofilm formation genes were determined by PCR. Biofilms were grown using flat-bottomed polystyrene tablets, followed by coloring, fixation, elution and detection. Population diversity was assessed by the multilocus sequence typing. About a quarter of cases of bacteremia and central nervous system infection caused by A. baumannii had an unfavorable outcome. Resistance to carbapenems, aminoglycosides, fluoroquinolones was more than 70%. Carbapenemases of the OXA-23 (24%) and OXA-40 (41%) groups were identified. The study of biofilm production showed that A. baumannii isolates formed biofilms of varying intensity: weak biofilms (59%), moderate (35%) and strong (6%). During determining the sensitivity to meropenem for biofilm and planktonic forms of cultures, it was determined that the minimum inhibitory concentrations of meropenem were significantly higher for biofilms than for planktonic forms. The minimum inhibitory concentrations of meropenem for plankton cells ranged from 0.5 to 512 mg/l. While in biofilms the same microorganisms had in vitro minimum inhibitory concentrations of meropenem within 128 to 512 mg/l and higher. All isolates bore biofilm formation regulating genes: bfmR, bap and katE. The ompA gene was found in 94% strains, and the csuA/B gene was found in 88%. The population pattern of A. baumannii isolated from blood and cerebrospinal fluid of children was represented by nine different sequence types. Most of the isolates were represented by genotypes: ST944Oxf, ST1550Oxf, ST1104Oxf belonging to the international clonal line ICL6, and ST450Oxf, ST2063Oxf and ST1102Oxf of the international clonal line ICL2. Blood-borne and central nervous system infections associated with A. baumannii have a great importance in clinical practice. This microorganism is able to persist for a long time on biotic and abiotic surfaces, has a wide natural and acquired antibiotics resistance.

Objective - assessment of RT-PCR for the detection of carbapenem-resistance genes in gram-negative bacteria. A total, 499 strains of gram-negative microorganisms isolated in two pediatric hospitals in 2019-2020 were studied. Species identification was performed using MALDI-ToF mass-spectrometry (Bruker Daltonics, Germany). Meropenem and imipenem minimal inhibitory concentration (MIC) was determined by E-test method (BioMerieux, France). The presence of acquired carbapenemase genes of IMP, NDM, VIM, KPC, OXA-48, OXA-23, OXA-40, OXA-58-groups was determined by RT-PCR. Klebsiella pneumoniae (34%), Escherichia coli (4%), Serratia marcescens (6%) and other members of Enterobacterales (6%), also gram-negative non-glucose-fermenting bacteria Acinetobacter baumannii (14%), Pseudomonas aeruginosa (36%) were found among selected strains. Carbapenemase production was found in 385 isolates (77%). The main mechanism determining carbapenem resistance in P. aeruginosa was the production of blaVIM (100%). A. baumanii strains harbored OXA-23 (55%) and OXA-40 (45%) carbapenemases. The major determinant of carbapenem resistance in K. pneumoniae isolates was OXA-48 carbapenemase, detected in 63% strains, 13% of the strains possessed blaNDM-group, 16% isolates had a combination of blaNDM-group and blaOXA-48-like. Carbapenemase of KPC-group was found in 8% K. pneumoniae strains. OXA-48 carbapenemase prevailed (95%) among S. marcescens strains. Most of E. coli isolates harbored metallo-beta-lactamase NDM (89%). Other members of Enterobacterales most often had OXA-48 carbapenemase (57%), 39% of the isolates carried blaNDM-group. In one strain, a combination of blaNDM-group and blaOXA-48-like was discovered. RT-PCR is a fast and reliable method for the detection of acquired carbapenemases and can be recommended for routine use in bacteriological laboratories.

Objective. To assess antimicrobial susceptibility, presence of resistance genes and determine the phenotypic groups of K. pneumoniae, P. aeruginosa and A. baumannii isolated from blood and cerebrospinal fluid of children with nosocomial infections in intensive care units from 2014 to 2020. Materials and Methods. Minimum inhibitory concentrations of antibiotics were determined using the serial broth microdilution method. The identification of genes encoding the production of carbapenemases was carried out using hybridization fluorescence detection. Results. A total of 63 isolates of K. рneumoniae, 23 isolates of P. aeruginosa and 14 isolates of A. baumannii were tested in this study. K. pneumoniae was detected in 10.3%. P. aeruginosa was isolated at a frequency of 3.5%. A. baumannii accounted for 2.3%. The proportion of carbapenemresistant K. pneumoniae strains to meropenem and imipenem was 33% and 37%, respectively, of all isolates. Resistance to colistin and polymyxin in K. pneumoniae isolates was 33% and 24%, respectively. The production of carbapenemases OXA-48 was detected in 25 (89%) isolates. The presence of NDM, VIM, KPC carbapenemases was not detected. Among P. aeruginosa, 65% were resistant to meropenem, and 74% to imipenem. The highest activity against P. aeruginosa in vitro was exhibited by polymyxins. There were no strains that were insensitive to colistin. The detection rate of metallo-β-lactamases (MBL) in P. aeruginosa strains was 48%. Only VIM-type MBLs were identified. No other types of MBL have been found. A. baumannii was non-susceptible to meropenem in 64% and to imipenem in 71%. The highest in vitro activity against A. baumannii was shown by polymyxin. Rate of colistin resistance was 29%. The OXA-40 and OXA-23 genes were detected in 5 and 3 isolates, respectively. Conclusions. There were high resistance rates to most antimicrobials among K. pneumoniae, P. aeruginosa и A. baumannii isolated from blood and cerebrospinal fluid in children with nosocomial infections. The increase in carbapenem resistance rates was also observed. Carbapenem resistance was due to OXA48 carbapenemases in K. pneumoniae, VIM-type MBLs in P. aeruginosa, and OXA-40 and OXA-23 carbapenemases in A. baumannii.